No abstract

An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD 50 ]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD 50 ) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD 50 ), and 117 pg/ml for BoNT/F (less than 1 LD 50 ) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.

Botulinum neurotoxin (BoNT), which cause the deadly neuroparalytic disease, botulism, is the most toxic substance known to man. BoNT can be used as potential bioterrorism agents, and therefore, pose great threat to national security and public health. Rapid and sensitive detection of BoNTs using molecular and biochemical techniques is an essential component in the diagnosis of botulism, and is yet to be achieved. The most sensitive and widely accepted assay method for BoNTs is mouse bioassay, which takes 4 days to complete. This clearly can not meet the need for clinical diagnosis of botulism, botulinum detection in field conditions, and screening of large scale samples. Consequently, the clinical diagnosis of botulism relies on the clinical symptom development, thus limiting the effectiveness of antitoxin treatment. In response to this critical need, many in vitro methods for BoNT detection are under development. This review is focused on recently developed in vitro detection methods for BoNTs, and emerging new technologies with potential for sensitive and rapid in vitro diagnostics for botulism.

No abstract

Current biodetection illumination technologies (laser, LED, tungsten lamp, etc.) are based on spot illumination with additional optics required when spatial excitation is required. Herein we describe a new approach of spatial illumination based on electroluminescence (EL) semiconductor strips available in several wavelengths, greatly simplifying the biosensor design by eliminating the need for additional optics. This work combines EL excitation with charge-coupled device (CCD) based detection (EL-CCD detector) of fluorescence for developing a simple portable detector for botulinum neurotoxin A (BoTN-A) activity analysis. A Förster Resonance Energy Transfer (FRET) activity assay for BoTN-A was used to both characterize and optimize the EL-CCD detector. The system consists of two modules: (1) the detection module which houses the CCD camera and emission filters, and (2) the excitation and sample module, containing the EL strip, the excitation filter and the 9-well sample chip. The FRET activity assay used in this study utilized a FITC/DABCYL-SNAP-25 peptide substrate in which cleavage of the substrate by BoTN-A, or its light chain derivative (LcA), produced an increase in fluorescence emission. EL-CCD detector measured limits of detection (LODs) were similar to those measured using a standard fluorescent plate reader with valves between 0.625 and 1.25 nM (31-62 ng/ml) for LcA and 0.313 nM (45 ng/ml) for the full toxin, BoTN-A. As far as the authors are aware this is the first demonstration of phosphor-based EL strips being used for the spatial illumination/excitation of a surface, coupled with CCD for point of care detection.

Chickpea is an important grain legume of the semi arid tropics and warm temperate zones, and forms one of the major components of human diet. However, a narrow genetic base of cultivated chickpea (Cicer arietinum L.) has hindered the progress in realizing high yield gains in breeding programs. Furthermore, various abiotic and biotic stresses are the major bottlenecks for increasing chickpea productivity. Systematic collection and evaluation of wild species for useful traits has revealed presence of a diverse gene pool for tolerance to the biotic and abiotic stresses. Relationships among the species of genus Cicer are presented based on crossability, karyotype and molecular markers. The reproductive barriers encountered during interspecific hybridization are also examined. Recent information on genetic linkage maps, comparison of isozymes

The suitability and sensitivity of two in vitro lateral-flow assays for detecting Clostridium botulinum neurotoxins (BoNTs) in an assortment of foods were evaluated. Toxin extraction and preparation methods for various liquid, solid, and high-fat-content foods were developed. The lateral-flow assays, one developed by the Naval Medical Research Center (Silver Spring, MD) and the other by Alexeter Technologies (Gaithersburg, MD), are based on the immunodetection of BoNT types A, B, and E. The assays were found to be rapid and easy to perform with minimum requirements for laboratory equipment or skills. They can readily detect 10 ng/ml of BoNT types A and B and 20 ng/ml of BoNT type E. Compared to other in vitro detection methods, these assays are less sensitive, and the assessment of a result is strictly qualitative. However, the assay was found to be simple to use and to require minimal training. The assays successfully detected BoNT types A, B, and E in a wide variety of foods, suggesting their potential usefulness as a preliminary screening system for triaging food samples with elevated BoNT levels in the event of a C. botulinum contamination event.

The emergence and dissemination of carbapenemases, bacterial enzymes able to inactivate most β-lactam antibiotics, in Enterobacteriaceae is of increasing concern. The concurrent spread of resistance against colistin, an antibiotic of last resort, further compounds this challenge further. Whole-genome sequencing (WGS) can play a significant role in the rapid and accurate detection/characterization of existing and emergent resistance determinants, an essential aspect of public health surveillance and response activities to combat the spread of antimicrobial resistant bacteria. In the current study, WGS data was used to characterize the genomic content of antimicrobial resistance genes, including those encoding carbapenemases, in 10 multidrug-resistant Klebsiella pneumoniae isolates from Pakistan. These clinical isolates represented five sequence types: ST11 (n = 3 isolates), ST14 (n = 3), ST15 (n = 1), ST101 (n = 2), and ST307 (n = 1). Resistance profiles against 25 clinically-relevant antimicrobials were determined by broth microdilution; resistant phenotypes were observed for at least 15 of the 25 antibiotics tested in all isolates except one. Specifically, 8/10 isolates were carbapenem-resistant and 7/10 isolates were colistin-resistant. The blaNDM-1 and blaOXA-48 carbapenemase genes were present in 7/10 and 5/10 isolates, respectively; including 2 isolates carrying both genes. No plasmid-mediated determinants for colistin resistance (e.g. mcr) were detected, but disruptions and mutations in chromosomal loci (i.e. mgrB and pmrB) previously reported to confer colistin resistance were observed. A blaOXA-48-carrying IncL/M-type plasmid was found in all blaOXA-48-positive isolates. The application of WGS to molecular epidemiology and surveillance studies, as exemplified here, will provide both a more complete understanding of the global distribution of MDR isolates and a robust surveillance tool useful for detecting emerging threats to public health.